X-ray Crystallography Research Articles

Mechanochemical synthesis of iron aluminyl complexes.

A series of iron aluminyl complexes have been synthesised in good crystalline yields from reactions between bulky diamido aluminium iodide complexes and K[Fe(CO)2Cp] in the solid state. The series of metal-metal bonded complexes have been characterised by X-ray crystallography and were investigated using density functional theory to probe the effects of ligand substitution on the Al-Fe bond.

Studies on the Coordination Patterns of Diamine-Bridged Tetraphenoxy Ligands with Group 3 and 4 Metals.

Alkane elimination reactions between the diamino- and dianilino-bridged tetrakis(phenolate) proligands 1a,b-H4 and precursors M(CH2SiMe3)3(THF)2, M(CH2C6H4-o-NMe2)3 (M = Sc and Y), and Hf(CH2Ph)4 were investigated. The diamino-bridged 1a-H4 afforded nonsymmetric complex 2a-Y2 incorporating two metal centers in different coordination environments. This one and other dinuclear compounds 2b-Sc2, 2a-Hf2, and 2b-Hf2 were characterized by NMR spectroscopy, elemental analysis, and X-ray diffraction study (for 2a-Y2 and 2b-Sc2) and turned out to be symmetric in solution. Compound 2a-Y2, upon treatment with 2 equiv of 2-phenylpyridine, afforded symmetric bis(aryl) product 3a-Y2, which was authenticated by NMR spectroscopy and X-ray crystallography. The mechanism of its formation was studied by DFT computations and presumably involves a cooperative reorganization process within the nonsymmetric parent 2a-Y2 to afford a symmetric isomer prior to its reaction with 2-phenylpyridine.

Synthesis and characterisation of copper(I) complexes with relevance to intramolecular Ullmann O,S-arylation.

Copper-catalysed intramolecular Ullman arylation has been frequently used to synthesise benzoxazoles and benzothiazoles. Despite widespread use, investigations into the mechanism and speciation of copper-containing complexes relevant to the catalytic pathway have remained relatively limited. Accordingly, this study aims to elucidate the structural details of potential copper(I) intermediates through the analysis of their solid-state structures using X-ray crystallography, while also investigating the reactivities of these complexes. Five novel copper complexes are reported which are formed prior to the aryl halide activation step and feature distinct aggregation modes based on either Cu4N4O4C4 or Cu4N4S4C4 clusters.

Targeting a microbiota Wolbachian aminoacyl-tRNA synthetase to block its pathogenic host.

The interplay between humans and their microbiome is crucial for various physiological processes, including nutrient absorption, immune defense, and maintaining homeostasis. Microbiome alterations can directly contribute to diseases or heighten their likelihood. This relationship extends beyond humans; microbiota play vital roles in other organisms, including eukaryotic pathogens causing severe diseases. Notably, Wolbachia, a bacterial microbiota, is essential for parasitic worms responsible for lymphatic filariasis and onchocerciasis, devastating human illnesses. Given the lack of rapid cures for these infections and the limitations of current treatments, new drugs are imperative. Here, we disrupt Wolbachia's symbiosis with pathogens using boron-based compounds targeting an unprecedented Wolbachia enzyme, leucyl-tRNA synthetase (LeuRS), effectively inhibiting its growth. Through a compound demonstrating anti-Wolbachia efficacy in infected cells, we use biophysical experiments and x-ray crystallography to elucidate the mechanism behind Wolbachia LeuRS inhibition. We reveal that these compounds form adenosine-based adducts inhibiting protein synthesis. Overall, our study underscores the potential of disrupting key microbiota to control infections.

Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Delocalized quinolinium-macrocyclic peptides, an atypical chemotype for CNS penetration.

Macrocyclic drugs can address an increasing range of molecular targets but enabling central nervous system (CNS) access to these drugs has been viewed as an intractable problem. We designed and synthesized a series of quinolinium-modified cyclosporine derivatives targeted to the mitochondrial cyclophilin D protein. Modification of the cation to enable greater delocalization was confirmed by x-ray crystallography of the cations. Critically, greater delocalization improved brain concentrations. Assessment of the compounds in preclinical assays and for pharmacokinetics identified a molecule JP1-138 with at least 20 times the brain levels of a non-delocalized compound or those reported for cyclosporine. Levels were maintained over 24 hours together with low hERG potential. The paradigm outlined here could have widespread utility in the treatment of CNS diseases.

Conformation of factor Xa in solution revealed by single molecule spectroscopy

All current X-ray structures of fXa are devoid of the γ-carboxyglutamate (Gla) domain and fail to reveal the overall conformation of the free protein. The recent cryo-EM structure of fXa in the prothrombinase complex is the only structure of full length fXa and shows that the Gla domain is positioned at an angle relative to the EGF1 domain. Establish if the curved conformation of fXa revealed by cryo-EM is also present in solution. The conformation of fXa in solution is studied by single molecule Förster resonance energy transfer (smFRET). The conformation of full length fXa in solution is resolved for the first time. The conformation is curved and extremely sensitive to Ca2+. It does not differ significantly from its zymogen form or from that present in the prothrombinase complex free or bound to the physiological substrates prothrombin and meizothrombin. Measurements by smFRET reveal that fXa has a curved conformation in solution, free or bound to physiological ligands, and validate the recent cryo-EM structures of prothrombinase. The drastic conformational changes observed in the absence of Ca2+ suggest that the structural architecture of fXa changes upon administration of vitamin-K antagonists that perturb the interaction of the Gla domain with divalent cations.

Crystal structure of the cytotoxic macrocyclic trichothecene Isororidin A.

The highly cytotoxic macrocyclic trichothecene Isororidin A (C29H40O9) was isolated from the fungus Myrothesium verrucaria endophytic on the wild medicinal plant `Datura' (Datura stramonium L.) and was characterized by one- (1D) and two-dimensional (2D) NMR spectroscopy. The three-dimensional structure of Isororidin A has been confirmed by X-ray crystallography at 0.81 Å resolution from crystals grown in the orthorhombic space group P212121, with one molecule per asymmetric unit. Isororidin A is the epimer of previously described (by X-ray crystallography) Roridin A at position C-13' of the macrocyclic ring.

Template-Directed Selective Photodimerization Reactions of 5-Arylpenta-2,4-dienoic Acids.

We developed an efficient method that enables selective photodimerization of 5-arylpenta-2,4-dienoic acids (i.e., vinylogous cinnamic acids). The use of 1,8-dihydroxynaphthalene as a template ensures proximity of the two reacting olefins so that irradiation of template-bound dienoic acids gives mono [2 + 2] cycloaddition products in good to excellent yields (up to 99%), as single regioisomers, and with high diastereoselectivities (dr = 3:1 to 13:1). The geometrical and stereochemical features of compounds 12a, 16a, and 22a were analyzed by X-ray crystallography.

Template-Directed Selective Photodimerization Reactions of 5-Arylpenta-2,4-dienoic Acids.

We developed an efficient method that enables selective photodimerization of 5-arylpenta-2,4-dienoic acids (i.e., vinylogous cinnamic acids). The use of 1,8-dihydroxynaphthalene as a template ensures proximity of the two reacting olefins so that irradiation of template-bound dienoic acids gives mono [2 + 2] cycloaddition products in good to excellent yields (up to 99%), as single regioisomers, and with high diastereoselectivities (dr = 3:1 to 13:1). The geometrical and stereochemical features of compounds 12a, 16a, and 22a were analyzed by X-ray crystallography.

Intramolecular [π4s + π4s] photocycloaddition of carbon- and nitrogen-bridged [32](1,4)naphthalenophanes.

[32](1,4)Naphthalenophanes, bearing carbon-bridge chains (syn- and anti-NPs) and nitrogen-bridge chains (syn- and anti-ANPs), were synthesized, and their X-ray structures and photoreactions were investigated. The intramolecular separation distance between the naphthalene cores for ANPs was shorter than that for NPs, suggesting that intramolecular interactions between the naphthalene rings were more efficient for ANPs compared to NPs. Upon photoirradiation at 300 nm, anti-NP, syn-ANP and anti-ANP produced the corresponding intramolecular [π4s + π4s] cycloadducts, whereas syn-NP gave an unidentified complex product mixture. Quantum yields for the photo-consumption (ΦPC) of NPs and ANPs were evaluated to quantitatively compare their photoreactivity. The ΦPC values of ANPs were approximately two-fold higher than those of ANPs.Noteworthily, the ΦPC value of syn-ANP was estimated to be unity. Based on these results we discuss the effects of the alignments of the naphthalene cores (anti vs. syn) and the bridging elements (C-bridge vs. N-bridge) on the photoreaction efficiencies of [32](1,4)naphthalenophanes.

Intramolecular [π4s + π4s] photocycloaddition of carbon- and nitrogen-bridged [32](1,4)naphthalenophanes.

[32](1,4)Naphthalenophanes, bearing carbon-bridge chains (syn- and anti-NPs) and nitrogen-bridge chains (syn- and anti-ANPs), were synthesized, and their X-ray structures and photoreactions were investigated. The intramolecular separation distance between the naphthalene cores for ANPs was shorter than that for NPs, suggesting that intramolecular interactions between the naphthalene rings were more efficient for ANPs compared to NPs. Upon photoirradiation at 300 nm, anti-NP, syn-ANP and anti-ANP produced the corresponding intramolecular [π4s + π4s] cycloadducts, whereas syn-NP gave an unidentified complex product mixture. Quantum yields for the photo-consumption (ΦPC) of NPs and ANPs were evaluated to quantitatively compare their photoreactivity. The ΦPC values of ANPs were approximately two-fold higher than those of ANPs.Noteworthily, the ΦPC value of syn-ANP was estimated to be unity. Based on these results we discuss the effects of the alignments of the naphthalene cores (anti vs. syn) and the bridging elements (C-bridge vs. N-bridge) on the photoreaction efficiencies of [32](1,4)naphthalenophanes.

An RNA aptamer exploits exosite-dependent allostery to achieve specific inhibition of coagulation factor IXa

Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.

Structure analysis of the telomere resolvase from the Lyme disease spirochete Borrelia garinii reveals functional divergence of its C-terminal domain.

Borrelia spirochetes are the causative agents of Lyme disease and relapsing fever, two of the most common tick-borne illnesses. A characteristic feature of these spirochetes is their highly segmented genomes which consists of a linear chromosome and a mixture of up to approximately 24 linear and circular extrachromosomal plasmids. The complexity of this genomic arrangement requires multiple strategies for efficient replication and partitioning during cell division, including the generation of hairpin ends found on linear replicons mediated by the essential enzyme ResT, a telomere resolvase. Using an integrative structural biology approach employing advanced modelling, circular dichroism, X-ray crystallography and small-angle X-ray scattering, we have generated high resolution structural data on ResT from B. garinii. Our data provides the first high-resolution structures of ResT from Borrelia spirochetes and revealed active site positioning in the catalytic domain. We also demonstrate that the C-terminal domain of ResT is required for both transesterification steps of telomere resolution, and is a requirement for DNA binding, distinguishing ResT from other telomere resolvases from phage and bacteria. These results advance our understanding of the molecular function of this essential enzyme involved in genome maintenance in Borrelia pathogens.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Relevance of G protein-coupled receptor (GPCR) dynamics for receptor activation, signalling bias and allosteric modulation.

G protein-coupled receptors (GPCRs) are one of the major drug targets. In recent years, computational drug design for GPCRs has mainly focused on static structures obtained through X-ray crystallography, cryogenic electron microscopy (cryo-EM) or in silico modelling as a starting point for virtual screening campaigns. However, GPCRs are highly flexible entities with the ability to adopt different conformational states that elicit different physiological responses. Including this knowledge in the drug discovery pipeline can help to tailor novel conformation-specific drugs with an improved therapeutic profile. In this review, we outline our current knowledge about GPCR dynamics that is relevant for receptor activation, signalling bias and allosteric modulation. Ultimately, we highlight new technological implementations such as time-resolved X-ray crystallography and cryo-EM as well as computational algorithms that can contribute to a more comprehensive understanding of receptor dynamics and its relevance for GPCR functionality.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

Well-Defined Highly-Coordinated Copper(III) Iodide and Pincer Tris(trifluoromethyl)copper Complexes.

Copper(III) iodide and bromide complexes representing a unique combination of highly-coordinated metal and soft polarizable anions were synthesized and fully characterized, including X-ray crystallography. Ligand substitution in well-defined highly-coordinated copper complex PyCu(CF3)3 with pincer ligands was achieved to give formally octahedral copper(III) complexes.

Well-Defined Highly-Coordinated Copper(III) Iodide and Pincer Tris(trifluoromethyl)copper Complexes.

Copper(III) iodide and bromide complexes representing a unique combination of highly-coordinated metal and soft polarizable anions were synthesized and fully characterized, including X-ray crystallography. Ligand substitution in well-defined highly-coordinated copper complex PyCu(CF3)3 with pincer ligands was achieved to give formally octahedral copper(III) complexes.